 This is Arne-Eloson and Anne Kauke and we will talk about our recent paper in Protein Science about aquapoint 1 folding. In our Protein Science paper we have studied unusual folding of aquapoint 1 and in particular how helices can rotate after it has been inserted into the membrane. Arne, what are the steps involved in membrane protein folding? Membrane protein folding is a two-stage process. First, these hydrophobic helices are inserted one by one to the membrane and then these helices fold together to a final structure. This sounds quite straightforward. Is it always such a simple process? The folding step is very complicated and they can take place a lot of changes even after the helices are inserted into the membrane. There can happen changes in topology, helices or even nearly half a protein can be inserted. And now there has been a lot of debate whether these changes can be placed spontaneously or whether they require specific machinery. Aquapoint 1 does not follow this standard two-step procedure of folding. How does it insert and folds? It is initially inserted as a complex intermediate and then during folding two of the helices are inserted and helix 3 rotates 180 degrees. On the other hand, its closed homolog aquapoint 4 is completely normal incisional. During folding of aquapoint 1, but not for aquapoint 4 which is the latest protein helix 3 has to be reverted in the membrane. So what is unique about these sequence features close to helix 3 in aquapoint 1 that cannot be observed in aquapoint 4? We noticed from hydrophobicity curve that there is in fact an extra peak just before helix 3 and there is just a low barrier between these two. What consequences do you think this extra peak has for aquapoint 1 folding? Perhaps this extra peak could shift to the membrane instead of helix 3 and if this would happen, then helix 3 would be ready to reinsert to the membrane in correct orientations together with aquapoint 4. Anni, you observed this peak from bifematic analysis. How did you proceed to show that this really could happen? We confirmed experimentally that this preceding peak really can insert to the membrane and that there is several alternative inserting segments at this region. That barrier is relatively low and our suggested shift seems feasible. In our model, helix 3 would be the initiation of a larger rescaling process that would involve helix 2 and 4 also. However, this would mean that you would need to bring polar residues across the membrane. Is this really energetically feasible? It should be remembered that there is quite much protein in the membrane so that would be hydrophobicity and make these sort of changes easier. Would these large scales rearrangement be possible without an external machinery? We believe that this shift would take place without specific machinery. Do you think the mechanism in aquapoint 1 folding is applicable to other membrane models also? I see our study as an example of what kind of mechanism these third of large scale rearrangements could use. What would you like to study next? It would be interesting to know how the rest of the rearrangement can take place and whether that requires any specific machinery. In this study, it will be interesting to see how common it is that these radical rearrangements can take place spontaneously because there exists such examples.